ASTM F1984-1999(2003) Standard Practice for Testing for Whole Complement Activation in Serum by Solid Materials《用固体材料测试血浆中整个活性的标准操作规程》.pdf

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1、Designation: F 1984 99 (Reapproved 2003)Standard Practice forTesting for Whole Complement Activation in Serum by SolidMaterials1This standard is issued under the fixed designation F 1984; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revis

2、ion, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This practice provides a protocol for rapid, in vitroscreening for whole complement activating prope

3、rties of solidmaterials used in the fabrication of medical devices that willcontact blood.1.2 This practice is intended to evaluate the acute in vitrowhole complement activating properties of solid materialsintended for use in contact with blood. For this practice, thewords “serum” and “complement”

4、are used interchangeably(most biological supply houses use these words synonymouslyin reference to serum used as a source of complement).1.3 This practice consists of two procedural parts. Proce-dure A describes exposure of solid materials to a standard lot ofhuman serum, using a 0.1-mL serum/13 x 1

5、00-mm disposabletest tube. Cellulose acetate powders and fibers are used asexamples of test materials. Procedure B describes assaying theexposed serum for significant functional whole complementdepletion as compared to control samples.1.4 This practice does not address function, elaboration, ordeple

6、tion of individual complement components, nor does itaddress the use of plasma as a source of complement.1.5 This practice is one of several developed for theassessment of the biocompatibility of materials. Practice F 748may provide guidance for the selection of appropriate methodsfor testing materi

7、als for other aspects of biocompatibility.2. Referenced Documents2.1 ASTM Standards:2F 748 Practice for Selecting Generic Biological Test Meth-ods for Materials and Devices2.2 ISO Document:ISO 10993-4: Biological Evaluation of Medical Devices,Part 4: Selection of Tests for Interactions with Blood33.

8、 Terminology3.1 Abbreviations:3.1.1 Abantibody (hemolysin).3.1.2 BBSbarbital buffered saline.3.1.3 BBS-Gbarbital buffered salinegelatin.3.1.4 BBS-GMbarbital buffered salinegelatin metals.3.1.5 C8complement.3.1.6 EDTAethylenediaminetetraacetic acid, disodiumsalt: dihydrate.3.1.7 HShuman serum.3.1.8 P

9、VDFpolyvinylidene fluoride.3.1.9 RBCred blood cell(s).4. Summary of Practice4.1 Solid material specimens are exposed to contact with astandard lot of complement under defined conditions (Proce-dure A). Exposed serum then is tested for significant functionalcomplement depletion compared to controls u

10、nder identicalconditions (Procedure B).5. Significance and Use5.1 Inappropriate activation of complement by blood-contacting medical devices may have serious acute or chroniceffects on the host. This practice is useful as a simple,inexpensive screening method for determining functionalwhole compleme

11、nt activation by solid materials in vitro.5.2 This practice is composed of two parts. In Part A(Section 11), human serum is exposed to a solid material.Complement may be depleted by the classical or alternativepathways. In principle, nonspecific binding of certain comple-ment components also may occ

12、ur. The alternative pathway candeplete later acting components common to both pathways,that is components other than C1, C4, and C3 (1).4In Part B(Section 12), complement activity remaining in the serum afterexposure to the test material is assayed by classical pathway-mediated lysis of sensitized R

13、BC.1This practice is under the jurisdiction of ASTM Committee F04 on Medical andSurgical Materials and Devices and is the direct responsibility of SubcommitteeF04.16 on Biocompatibility Test Methods.Current edition approved Nov. 1, 2003. Published December 2003. Originallyapproved in 1999. Last prev

14、ious edition approved in 1999 as F 1984 99.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Available from Am

15、erican National Standards Institute (ANSI), 25 W. 43rd St.,4th Floor, New York, NY 10036.4The boldface numbers in parentheses refer to the list of references at the end ofthis specification.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United St

16、ates.5.3 Assessment of in vitro whole complement activation, asdescribed here, provides one method for predicting potentialcomplement activation by medical materials intended forclinical application in humans when the material contacts theblood. Other test methods for complement activation areavaila

17、ble, including assays for specific complement compo-nents and their split products (see X1.3 and X1.4).5.4 This in vitro test method is suitable for adoption inspecifications and standards for screening solid materials foruse in the construction of medical devices intended to beimplanted in the huma

18、n body or placed in contact with humanblood.6. Preparation of Buffers6.1 Buffers, are prepared according to detailed protocol (2).“Water” refers throughout to distilled, endotoxin-free water.The use of barbital (veronal) buffer is recommended. Barbitalis a class IV regulated substance and requires a

19、 DEA (3) licensefor purchase. The use of other buffer systems, such as, TRIS, ispermissible if they have been demonstrated not to activatecomplement(4).6.2 5X Stock BBS (barbital-buffered saline), is prepared byadding 20.75 g NaCl plus 2.545 g sodium barbital (sodium-5,5-diethyl barbiturate) to abou

20、t 400 mL water. The pH isadjusted to 7.35 with 1 N HCl, then brought to a final volumeof 500 mL in a volumetric flask.6.3 Metals Solution, is prepared by making a 2.0 M solutionof MgCl2(40.66 g MgCl26H2O into 100 mL distilledendotoxin-free water), and a 0.3 M solution of CaCl2(4.41 gCaCl22H2O into 1

21、00 mL distilled endotoxin-free water), andcombining the two solutions 1:1 (v:v). These solutions arestable one month at 4C.6.4 BBS-GM Working Solution, is prepared daily, by dis-solving 0.25 g gelatin in 50 mL endotoxin-free distilled waterthat is gently heated and stirred. The gelatin solution is a

22、ddedto 50 mL 5X stock BBS plus 0.25 mL metals solution, broughtup to about 200 mL, then adjusted to pH 7.35 (with1NHClor 1 N NaOH) before bringing the final volume to 250 mL ina volumetric flask.6.5 BBS-G Working Solution, is prepared the same way, butthe addition of the metals solution is omitted.6

23、.6 10X Stock EDTA (0.1 M disodium dihydrate EDTA),isprepared by adding 7.44 g disodium EDTA2 H2O to about 160mL water, adjusting the pH to 7.65 (with 1 N NaOH or 1 NHCl), then bringing the volume to 200 mL in a volumetricflask.6.7 BBS-G-EDTA (to be used in preparing RBC beforebeing washed out), is p

24、repared by adding 10 mL of stock 10XEDTA to 90 mL of BBS-G in a volumetric flask.7. Preparation of Sheep RBC7.1 Commercially-obtained sheep red blood cells (RBC)preserved in Alsevers solution are stored at 4C. The cells arediscarded after eight weeks or when the supernatant from thesecond wash conta

25、ins hemoglobin by visual inspection.NOTE 1All centrifugations are at 4C. Except where indicated, allreagents, tubes, and cell preparations are kept on ice.7.2 Five mL of sheep RBC are centrifuged at 1 000xgfor10 min.7.3 The cell pellet is resuspended in 10 mL of coldBBS-G-EDTA and incubated for 10 m

26、in at 37C. The cells arecentrifuged, and the pellet resuspended in 10 mL of BBS-G-EDTA.7.4 The cells are centrifuged, the supernatant discarded(first wash), and the pellet resuspended in 10 mL of coldBBS-GM. Repeat twice (total of three washes).7.5 Adjust cell count spectrophotometrically (where ana

27、bsorbance of 0.56 corresponds to 1.5 x 108sheep RBC/mL, ata wavelength of 412 nm and a 1.0-cm light path for 1 volumeof cells in BBS-GM plus 24 volumes of water) or count witha hemocytometer, preparing 10 mL of 1.5 x 108cells/mL incold BBS-GM.7.6 The washed, diluted RBC can be held on ice and usedfo

28、r at least 12 h.8. Absorption of Serum (Complement)8.1 The use of human complement is required since thereare species differences in the efficiency of complement activa-tion and the test materials are to be used in humans. Humanserum suitable as a source of complement may be purchasedfrom biological

29、 supply houses, and generally, is labeled asreagent-grade complement.8.2 Human serum may be absorbed with sheep RBC inorder to remove naturally-occurring anti-sheep RBC hemolyticantibodies, though for most purposes, the amount of hetero-phile antibody in human serum is not enough to influence therea

30、ction assuming the cells are optimally sensitized withhemolysin. The procedure is detailed in 8.3-8.8.8.3 Fresh human serum or a commercial lot of human serumis obtained and stored at 70C. Fresh serum is preferred aslyophilized complement often is not as active as fresh serum.8.4 The serum is thawed

31、 on ice or reconstituted (if lyo-philized) with ice-cold (4C) distilled endotoxin-free water.8.5 All manipulations are done on ice, with ice cold reagentsand cells; centrifugations are carried out at 1000xgat4C. Itis critical that this entire procedure be done in the cold to avoidactivation of compl

32、ement in this step.8.6 Cold serum and cold, packed, washed sheep RBC aremixed, 0.1 mL RBC/2.5 mL serum, incubated for 10 min onice, then centrifuged at 1 000 x g for 10 min at 4C. Thesupernatant is transferred carefully to a new container on ice.8.7 The procedure in 8.6 is repeated twice.8.8 The abs

33、orbed human serum is stored in 0.51.0-mLaliquots (convenient for one-experiment use), in cold snap-capmicrofuge tubes and kept at 70C until used. Aliquots shouldbe thawed on ice, used on the day of thawing, and not berefrozen.9. Determination of Optimal Hemolysin Concentration9.1 Determination of op

34、timal hemolysin concentration isnecessary in order to conserve expensive reagents and to avoidprozone effects. Commercial rabbit anti-sheep RBC serum(Hemolysin) is obtained, thawed, or, if lyophilized, reconsti-tuted with distilled endotoxin-free water), heat-inactivated at56C for 30 min to inactiva

35、te the rabbit complement, aliquotedin convenient volumes, and stored at 70C until used.F 1984 99 (2003)29.2 To cold 13 x 100 mm disposable glass tubes, placed ina rack in an ice-bath, 0.1 mL of washed sheep RBC at 1.5 x 108cells/mL is added. If statistical evaluation of the results isdesired, three

36、replicate tubes for each condition should be used.Otherwise, duplicates or even single dilution tubes are suffi-cient. One set of three replicate tubes receives only 0.1 mL ofcold BBS-GM/tube (no RBC control, for complement color).9.3 To the RBC-containing tubes, one set of three tubes gets1.1 mL co

37、ld distilled H2O/tube (total lysis control), anothergets 0.1 mL BBS-GM (no hemolysin control), and the othersets get 0.1 mL each of 1:2 serial dilutions of hemolysin (tests).Dilutions between 1:400 to 1:25 600 antibody are recom-mended, with two sets of 1:400. The no RBC control receives0.1 mL of ad

38、ditional BBS-GM.9.4 Each tube is mixed quickly by gentle shaking toresuspend cells, the rack is placed in a 37C water bath,incubated 10 min, then returned to the ice bath.9.5 One of the two sets of 1:400 antibody gets 1.0 mL ofcold BBS-GM (no-complement control). All other tubes be-sides the total l

39、ysis control set get 0.5 mL cold BBS-GM, then0.5 mL of absorbed human serum (complement) diluted 1:100or 1:200.NOTE 2For a particular lot of human serum, a 1:100 or 1:200 dilutionshould provide sufficient complement activity. Also, percent lysis in theno-hemolysin (complement only) control should no

40、t exceed 10 %. If lysiswith the 1:100 dilution of complement exceeds 10 %, use 1:200. If theno-hemolysin control still exceeds 10 %, a different lot of serum will needto be tested.9.6 Tubes are shaken manually to suspend cells, then therack is incubated in a 37C water bath for 1h, and intermit-tentl

41、y shaken to keep cells in suspension.9.7 At the end of 1h, the rack is placed on ice. The coldtubes then are centrifuged at 1 000 x g for 10 min at 4C, andthe supernatants decanted to correspondingly numbered 13 x100-mm glass tubes.9.8 Absorbance of the supernatants is measured at 412 nm.Percent lys

42、is is calculated for each test and control tube bysubtracting from the 412 nm absorbance the no RBC control(mean of the three replicate tubes), dividing by the total lysiscontrol value (mean of the three replicate tubes), and multi-plying by 100.% lysis 5test absorbance 2 no RBC control absorbanceto

43、tal lysis absorbance3 100(1)9.9 Final % lysis for each condition is expressed as mean 6standard deviation of the three % lysis values for each three-replicate set.9.10 A titration curve is obtained by plotting the inverse ofthe hemolysin concentration versus % specific lysis. Twice theconcentration

44、of hemolysin that is just on the plateau of thetitration curve is used for sensitizing RBC for subsequentassays (optimal hemolysin concentration). Hemolysin isfreshly diluted from stock each day of use.10. Whole Complement Titration to Determine OptimalSerum Dilution10.1 If statistical evaluation of

45、 results is desired, all condi-tions should be assayed in triplicate, using three 13 x 100disposable glass test tubes per condition. Otherwise, duplicatesor single tubes are sufficient. Tubes are numbered in advance.Conditions include total lysis, no complement (no C), tests(dilutions of human serum

46、HS) with and without hemolysin,and no RBC (complement color control, at highest concentra-tion of serum used). All reagents, tubes, and manipulations aredone ice-cold, with tubes held in a rack in an ice slurry.10.2 Washed RBC are added to all tubes except no RBCtubes (0.1 mL/tube of a 1.5 x 108cell

47、s/mL suspension). NoRBC tubes get 0.1 mL cold buffer.10.3 Total lysis tubes get 1.1 mL distilled H2O. The no Cand test with hemolysin tubes get 0.1 mL optimal hemolysin(see 9.10), and no RBC tubes get 0.1 mL cold BBS-GM. Alltubes are shaken to resuspend cells, incubated in a 37C waterbath for 10 min

48、, and placed back on ice.NOTE 3Another acceptable procedure is to make up one large batchof hemolysin-sensitized erythrocytes to cover all the tests planned withinone weeks time. These cells are made up at5x108/mL and are stored at4C. They are washed each time they are used, and if hemolysis occurs,

49、new sensitized cells are prepared. These sensitized cells are ready to use,making the addition of hemolysin to each tube unnecessary, whichsimplifies the experiment. Unsensitized RBC can be used as controls fornonspecific lysis.10.4 To all but the total lysis tubes, a maximum volume of1.0 mL of cold BBS-GM is added, reduced by the amount ofdiluted serum (see 10.5), which will be added at a maximum0.5 mL volume. The no C tubes get 1.0 mL BBS-GM.10.5 The cold serum is diluted in cold BBS-GM to thedesired concentration (with minimal agitation).